Orchiectomy upregulates free soluble RANKL in bone marrow of aged rats☆
Introduction
Declining bioavailable sex steroid levels are a major risk factor for osteoporosis in men [1], [2]. The important role of androgens in the maintenance of an adult male skeleton is documented by the fact that withdrawal of androgens by orchiectomy (ORX) or androgen blockade induces high turnover osteopenia in humans, aged mice, and aged rats [3], [4], [5], [6], [7], [8], [9], [10], [11]. Although still a matter of debate, several lines of evidence from clinical and experimental studies suggest that the suppressive effect of androgens on cancellous and endocortical bone turnover may be mediated indirectly via aromatization of androgens into estrogens [2], [9], [11], [12], [13], [14].
The aged ORX rat model has been very well described in terms of its phenotype [8], [9], [11]. However, the molecular pathogenesis of androgen deficiency-induced bone loss is still poorly understood. The OPG/RANKL system has been proposed as a potential mediator of sex steroid deficiency-induced bone loss. RANKL has been identified as an essential cytokine for osteoclast differentiation and activation [15], [16], and OPG represents its naturally occurring inhibitor [17]. RANKL is produced by cells of the stromal cell lineage, activated T lymphocytes, but also B lymphocytes [16]. The OPG/RANKL ratio is an essential determinant of osteoclast cell biology and bone resorption [18], [19]. Sex steroids regulate the expression of OPG and RANKL in vitro [19]. Orchiectomy of male rats increased RANKL mRNA expression in bone [20], and conditional ablation of the androgen receptor (AR) in mice upregulated osteoblast RANKL mRNA expression [21]. Vice versa, estradiol decreased the OPG/RANKL mRNA ratio and increased trabecular bone mineral density (BMD) in vertebrae of male ORX mice [22]. A recent flow cytometric analysis of bone marrow aspirates demonstrated that estrogen deficiency in women upregulates the membrane expression of RANKL protein on alkaline phosphatase-positive osteoblastic cells, CD3-positive T cells, and CD20-positive B cells [23]. Similarly, in men who underwent suppression of sex steroids using a gonadotropin-releasing hormone agonist in combination with an aromatase inhibitor, a non-significant trend for increased RANKL mRNA levels was observed in alkaline phosphatase-positive osteoblastic cells obtained from bone marrow aspirates [24]. However, unambiguous clinical or experimental evidence linking alterations in the OPG/RANKL axis with androgen withdrawal-induced bone loss in vivo is lacking.
It was the aim of this study to investigate further the role of the OPG/RANKL system in ORX-induced osteopenia in aged rats. So far, the evaluation of the OPG/RANKL axis in rats has been hampered by the fact that there is no commercially available immunoassay for rat OPG. To circumvent the problem that OPG cannot be measured at the protein level in rats, we used a novel rat- and mouse-specific immunoassay to assess free sRANKL in serum and bone marrow of aged ORX rats. Free sRANKL is the bioactive form of sRANKL that is not bound to OPG and can access its receptor RANK. Free sRANKL is either produced by immune cells as a primary secreted form [16], or results from shedding of membrane-bound RANKL by matrix metalloproteinase 14 (MMP-14) or by a disintegrin and metalloproteinase (ADAM) 10 [25]. Although the physiological function of RANKL shedding from osteoblastic cells is still controversial, free sRANKL may be the critical determinant of osteoclast activation and survival, and it accurately reflected the state of bone resorption in OPG deficient mice [26]. In the current study, we tested the hypothesis that orchiectomy may upregulate free sRANKL in the bone microenvironment of aged rats.
Section snippets
Animal procedures
All animal procedures were approved by the Ethical Committee of the University of Munich and the local government authorities. Thirty-three 9-month-old male Fischer-344 rats (Charles River) weighing about 400 g were used for this experiment. Eight rats served as baseline controls. The remaining rats were either bilaterally ORX under ether anesthesia or sham-operated (SHAM, skin incision at the scrotum). The animals were kept in pairs at 24 °C with a 12 h/12 h light/dark cycle. All rats received
Results
As expected, seminal vesicle weight was profoundly reduced in ORX relative to SHAM controls, 2 months post-ORX (Fig. 1A). In agreement with our earlier results [11], [31], weekly subcutaneous injection of 6 mg/kg T in ricinus oil-benzyl benzoate normalized seminal vesicle organ weight (Fig. 1A), showing that this is a physiological dose of testosterone. pQCT analysis of the tibiae revealed reduced trabecular BMD at the proximal tibia (Fig. 1B), and reduced cortical thickness of the tibial shaft
Discussion
In accordance with previous data from our laboratory and other groups, our study has shown that androgen withdrawal induces high turnover cancellous and cortical bone osteopenia in aged, nongrowing rats, 2 months post-ORX [3], [4], [5], [6], [7], [8], [9], [10], [11]. The 2-month time point in the current experiment was chosen on the basis that the increase in bone turnover in aged ORX rats peaks 2 months after surgery [9]. Supplementation of ORX rats with T at a physiological dose of
Acknowledgments
This research was supported by a grant from Deutsche Forschungsgemeinschaft to RGE (ER 223/1–3). The authors wish to thank Claudia Bergow and Sieglinde Hirmer for excellent technical assistance. Testosterone undecanoate was a gift of Dr. Uwe Kollenkirchen (Bayer-Schering, Berlin, Germany). The free sRANKL assays were a gift of Dr. Gerhard Hawa (Biomedica Medizinprodukte GmbH and Co KG, Divischgasse 4, 1210 Vienna, Austria).
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Sources of funding: Deutsche Forschungsgemeinschaft ER223/1–3.